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BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 173888 DESCRIPTION OF THE PROBLEM no band. but other lot well work. SAMPLE human, HEK293-OATP1B3 transfected cells. PRIMARY ANTIBODY Mouse monoclonal [MDQ] to OATP2 (ab15442), Lot No.205852., Abcam 1/200 dilution overnight incubate DETECTION METHOD WesternBreeze chemiluminescent western blot immunodetection kit POSITIVE AND NEGATIVE CONTROLS USED positive control not work ANTIBODY STORAGE CONDITIONS 4 degree for 2 weeks SAMPLE PREPARATION used ProteoExtract, Native membrane protein extraction kit(Calbiochem) AMOUNT OF PROTEIN LOADED 20mg protein/lane ELECTROPHORESIS/GEL CONDITIONS NuPAGE Bis-Tris-Gel, reducing gel, 10%(Invitrogen) TRANSFER AND BLOCKING CONDITIONS WesternBreeze chemiluminescent western blot immunodetection kit SECONDARY ANTIBODY WesternBreeze chemiluminescent western blot immunodetection kit HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 15 DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? No. only antibody new one
Asked on Oct 04 2006
Thank you for your enquiry. I am sorry to hear that you have been having difficulties with this antibody. I have read through your technical questionaire and I have a few comments. OATP2 antibody [MDQ] (ab15442) has been tested for its applications of immunofluorescence, immunohistochemistry (Frozen sections) and immunoprecipitation. To our knowledge we are not aware of how this antibody behaves in western blotting. Therefore unfortunately we cannot guarantee it for this purpose. I am sorry to hear that you have observed differences between the batches of antibody that you have used. This is a monoclonal antibody and I would not expect different antibody preparations to have significant variation in their quality. However, occasional batch to batch variation does exist. Therefore given this I would like to recommend that you perform the optimisation to your protocol; Firstly I would like to suggest that you verify the integrity of your sample and preparation by performing a loading control immunolabelling using an antibody that targets a housekeeping protein such as GAPDH or alpha tubulin. Following on I would like to suggest that you significantly reduce the dilution of the antibody to ~1:20 in view of the antibody dilutions recommended for immunofluorescence and perform an overnight incubation at 4oC using BSA as a blocking agent. This serves to improve the sensitivity of the antibody.
Answered on Oct 06 2006